In vitro evolution of nucleic acids and encoded polypeptide

ABSTRACT

This invention relates to methods and reagents for selecting a desired protein or nucleic acid molecule by linking mRNA, with known or unknown sequences, to its translated protein to form a cognate pair. The cognate pair is selected based upon desired properties of the protein or the nucleic acid. This method also includes the evolution of a desired protein or nucleic acid molecule by amplifying the nucleic acid portion of the selected cognate pair, introducing variation into the nucleic acid, translating the nucleic acid, attaching the nucleic acid to its protein to form a second cognate pair, and re-selecting this cognate pair based upon desired properties.

RELATED APPLICATIONS

This application claims the benefit from provisional application U.S.Ser. No. 60/206,016, filed May 19, 2000, and is a continuation of U.S.patent application Ser. No. 09/859,809, filed May 17, 2001, now U.S.Pat. No. 6,962,781 the entirety of which is incorporated by referenceherein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions and methods for theselection of nucleic acids and polypeptides.

2. Description of the Related Art

Ligand-receptor interactions are of interest for many reasons, fromelucidating basic biological site recognition mechanisms to drugscreening and rational drug design. It has been possible for many yearsto drive in vitro evolution of nucleic acids by selecting molecules outof large populations that preferentially bind to a selected target, thenamplifying and mutating them for subsequent re-selection (Tuerk andGold, Science 249:505 (1990), herein incorporated by reference).

The ability to perform the selection process with proteins would beextremely useful. This would permit in vitro design and production ofproteins that bind specifically to chosen ligands. The use of proteins,as compared to nucleic acids, is particularly advantageous because thetwenty diverse amino acid side chains in proteins have far more bindingpossibilities than the four similar chains in nucleic acid side.Further, many biologically and medically relevant ligands bind proteins.

Both nucleic acid and protein evolution methods require access to alarge and highly varied population of test molecules, a way to selectmembers of the population that exhibit the desired properties, and theability to reproduce the selected molecules with mutated variations toobtain another large population for subsequent selection.

Prior attempts to develop a protein evolution method were primarilylimited by the inability of the proteins to reproduce themselves and theinability to link a polypeptide's encoding mRNA with the translatedproduct. Additionally, the generation of large peptide libraries andscreening methods have, until recently, required that the process havean in vivo expression step. Examples include yeast two- or three-hybrid,yeast display and phage display methods (Fields and Song, Nature 340:245(1989); Licitra and Liu, PNAS 93:12817 (1996); Boder and Wittrup, NatBiotechnol 15:553 (1997); and Scott and Smith, Science 249:386 (1990)).In vivo methods suffer from various disadvantages, including a limitedlibrary size and cumbersome screening steps. Additionally, undesiredselective pressures can be placed on the generation of variants bycellular constraints of the host.

In vitro methods have been developed more recently, using prokaryoticand eukaryotic in vitro translation systems, such as ribosome display(Mattheakis et al., PNAS 91:9022 (1994); Hanes and Plückthun, PNAS94:4937 (1997); Jermutus et al., Current Opinion in Biotechnology 9:534(1998), all herein incorporated by reference). These methods link theprotein and its encoding mRNA with the ribosome and the entire complexis screened against a ligand of choice. Potential disadvantages of thismethod include the large size of the ribosome, which could interferewith the screening of the attached, and relatively tiny, protein.

In 1997, two groups of workers developed an in vitro method of attachinga protein to its coding sequence during translation by using theribosomal peptidyl transferase with puromycin attached to a linker DNA(Szostak et al., International Patent Publication WO 98/31700; Robertsand Szostak PNAS 94:12297 (1997); Nemoto et al., FEBS Letters 414:405(1997), all herein incorporated by reference). Once the coding sequenceand peptides are linked, the peptides are exposed to a selected ligand.Selection or binding of the peptide by the ligand also selects theattached coding sequence, which can then be reproduced by standardmeans. Both Roberts and Szostak and Nemoto et al. used the technique ofattaching a puromycin molecule to the 3′ end of a coding sequence by aDNA linker or other non-translatable chain. Puromycin is a tRNA acceptorstem analog which accepts the nascent peptide chain under the action ofthe ribosomal peptidyl transferase and binds it stably and irreversibly,thereby halting translation. These methods suffer from certaindisadvantages. For example, the coding sequence encoding each peptidemust be known and be modified both initially and between each selection.Thus, the methods of Roberts and Nemoto cannot be used to select nativeunknown mRNAs. Further, the modification of the coding sequence addsseveral steps to the process. Finally, the attached puromycin on thelinker molecules may compete in the translation reaction with the nativetRNAs for the A site on the ribosome reading its coding sequence or anearby ribosome, and could thus “poison” the translation process, justas would unattached puromycin in the translation reaction solution.Inadvertent interactions between puromycin and ribosomes could result intwo kinds of reaction non-specificity: prematurely shortened proteinsand proteins attached to the wrong message. There are reports in theprior art that indicate that the avidity of the A site and the peptidyltransferase for the puromycin may be modulated by Mg⁺⁺ concentration(Roberts, Curr. Opin. Chem. Biol. 3:268 (1999), herein incorporated byreference). Although Mg⁺⁺ concentration may be titrated to control forthe first kind of non-specificity, i.e. premature termination oftranslation, it will not affect the second type, i.e. inaccuratemRNA-protein linkage.

Thus, a need exists for an in vitro nucleic acid-based protein evolutionsystem which does not necessarily require initial knowledge of thenucleic acid's sequence or repeated chemical modification of the nucleicacids, and which can accurately link a mRNA to its protein.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods to select andevolve desired properties of proteins and nucleic acids. In variousembodiments, the current invention provides modified tRNA's and tRNAanalogs. Other embodiments include methods for generating polypeptides,assays enabling selection of individual members of the population ofpolypeptides having desired characteristics, methods for amplifying thenucleic acids encoding such selected polypeptides, and methods forgenerating new variants to screen for enhanced properties.

In several embodiments, the present invention permits the attachment ofa protein to its message without requiring modification of native mRNA,although modified mRNA may still be used. The specificity of the methodsembodied in various aspects of the current invention are determined bythe specificity of the codon-anticodon interaction.

In a preferred embodiment, the invention permits the selection ofnucleic acids by selecting the proteins for which they code. This may beaccomplished by connecting the protein to its cognate mRNA at the end oftranslation, which in turn is done by connecting both the protein andmRNA to a tRNA or tRNA analog.

A preferred embodiment of the invention includes a tRNA molecule capableof covalently linking a nucleic acid encoding a polypeptide and thepolypeptide to the tRNA, wherein the linkage of the nucleic acid occurson a portion of the tRNA other than the linkage of the polypeptide, thetRNA comprising a linking molecule associated with the anticodon of thetRNA. Preferably, an amino acid or amino acid analog is attached to the3′ end of a tRNA molecule by a stable bond to generate a stableaminoacyl tRNA analog (SATA).

Other embodiments include a mRNA comprising a psoralen, preferablylocated in the 3′ region of the reading frame, more preferably at themost 3′ codon of the reading frame, most preferably at the 3′ stop codonof the reading frame. In preferred embodiments, linkage between the tRNAand the mRNA is a cross-linked psoralen molecule, more preferably afuran-sided psoralen monoadduct.

Several embodiments of the present invention include a method of stablylinking a nucleic acid, a tRNA, and a polypeptide encoded by the mRNAtogether to form a linked mRNA-polypeptide complex. In a preferredembodiment, the nucleic acid is an mRNA. The method can further compriseproviding a plurality of distinct nucleic acid-polypeptide complexes,providing a ligand with a desired binding characteristic, contacting thecomplexes with the ligand, removing unbound complexes, and recoveringcomplexes bound to the ligand.

Several methods of the current invention involve the evolution ofnucleic acid molecules and/or proteins. In one embodiment, thisinvention comprises amplifying the nucleic acid component of therecovered complexes and introducing variation to the sequence of thenucleic acids. In other embodiments, the method further comprisestranslating polypeptides from the amplified and varied nucleic acids,linking them together using tRNA, and contacting them with the ligand toselect another new population of bound complexes. Several embodiments ofthe present invention use selected protein-mRNA complexes in a processof in vitro evolution, especially the iterative process in which theselected mRNA is reproduced with variation, translated and againconnected to cognate protein for selection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates schematically one example of the complex formed bythe mRNA and its protein product when linked by a modified tRNA oranalog. As shown, a codon of the mRNA pairs with the anticodon of amodified tRNA and is covalently crosslinked to a psoralen monoadduct byUV irradiation. The translated polypeptide is linked to the modifiedtRNA via the ribosomal peptidyl transferase. Both linkages occur whilethe mRNA and nascent protein are held in place by the ribosome.

FIG. 2 illustrates schematically an example of the in vitro selectionand evolution process, wherein the starting nucleic acids and theirprotein products are linked (e.g., according to FIG. 1) and are selectedby a particular characteristic exhibited by the protein. Proteins notexhibiting the particular characteristic are discarded and those havingthe characteristic are amplified with variation, preferably viaamplification with variation of the mRNA, to form a new population. Invarious embodiments, nonbinding proteins will be selected. The newpopulation is translated and linked via a modified tRNA or analog, andthe selection process is repeated. As many selection andamplification/mutation rounds as desired can be performed to optimizethe protein product.

FIG. 3 illustrates one method of construction of a tRNA molecule of theinvention. In this embodiment, the 5′ end of a tRNA, a nucleic acidencoding an anticodon loop and having a molecule capable of stablylinking to mRNA (here, psoralen), and the 3′ end of tRNA modified with aterminal puromycin molecule are ligated to form a complete modified tRNAfor use in the in vitro evolution methods of the invention.

FIG. 4 describes two alternative embodiments by which the crosslinkingmolecule psoralen can be positioned to be capable of linking the mRNAwith the tRNA in the methods of the invention. A first embodimentincludes linking the psoralen monoadduct to the mRNA, and a secondembodiment includes linking the psoralen to the anticodon of the tRNA.Psoralen can either be monoadducted to the anticodon or the 3′ terminalcodon of the reading frame for known or partially known messages. Thiscan be done in a separate procedure from translation, i.e. beforetranslation occurs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Various aspects of the present invention use a tRNA mechanism that linksmessenger RNA (mRNA) to its translated protein product, forming a“cognate pair.” In several embodiments, mRNAs whose sequence is notknown can be expressed, its protein characterized through a selectionprocess against a ligand with desired or selected properties, andnucleic acid evolution—resulting in protein evolution—can be performedin vitro to arrive at molecules with enhanced properties. The cognatepairs are preferably attached via a linking tRNA, modified tRNA, or tRNAanalog. In a preferred embodiment, the tRNA is connected to the nascentpeptide by the ribosomal peptidyl transferase and to the mRNA through anultraviolet induced cross link between the anticodon of the tRNA or tRNAanalog and the codon of the RNA message. This can be done by, forexample thiouracil, but in a preferred method, the linker is a psoralencrosslink made from a psoralen monoadduct pre-placed on either the mRNAor preferably on the tRNA anticodon of choice. Preferably, a tRNA stopanticodon is selected. A stop codon/anticodon pair selects for fulllength transcripts. One skilled in the art will understand that a mRNAnot having a stop codon may also be used and that any codon or nucleicacid triplet may be used. A tRNA having an anticodon which is notnaturally occurring can be synthesized according to methods known in theart (e.g. FIG. 3).

The terms “protein,” “peptide,” and “polypeptide” are defined herein tomean a polymeric molecule of two or more units comprised of amino acidsin any form (e.g., D- or L-amino acids, synthetic or modified aminoacids capable of polymerizing via peptide bonds, etc.), and these termsmay be used interchangeably herein.

The term “pseudo stop codon” is defined herein to mean a codon which,while not naturally a nonsense codon, prevents a message from beingfurther translated. A pseudo stop codon may be created by using a“stable aminoacyl tRNA analog” or SATA, as described below. In thismanner, a pseudo stop codon is a codon which is recognized by and bindsto a SATA. Another method by which to create a pseudo stop codon is tocreate an artificial system in which the necessary tRNA having ananticodon complementary to the pseudocodon is substantially depleted.Accordingly, translation will stop when the absent tRNA is required,i.e. at the pseudo stop codon. One skilled in the art will appreciatethat are numerous ways to create a pseudo stop codon as defined herein.

The formation of connections between mRNA and its protein productgenerally requires a tRNA or tRNA analog with certain characteristics.In several embodiments of the current invention, the tRNA or tRNA analogwill have a stable peptide acceptor. This modification changes the tRNAor tRNA analog such that after it accepts the nascent peptide chain bythe action of the ribosomal peptidyl transferase, it holds the chain ina stable manner such that the peptidyl transferase cannot detach it.This may be accomplished by using a bond such as a 2′ ester on a 3′deoxy adenosine or an amino “acyl tRNA_(ox-red)” which can bind to theribosome, accept the peptide chain, and then not act as a donor in thenext transpeptidation (Chinali et al., Biochem. 13:3001 (1974);Krayevsky and Kukhanova, Prog. Nuc. Acid Res 23:1 (1979) and Sprinzl andCramer Prog. Nuc. Acid Res 22:1 (1979), all herein incorporated byreference).

In a preferred embodiment, an amino acid or amino acid analog isattached to the 3′ end of the tRNA or tRNA analog by a stable bond. Thisstable bond contrasts the labile, high energy ester bond that connectsthese two in the native structure. The stable bond not only protects thebond from the action of the peptidyl transferase, but also preserves thestructure during subsequent steps. For convenience, this modified tRNAor tRNA analog will be referred to as a “stable aminoacyl tRNA analog”or SATA. As used herein, a SATA is an entity which can recognize aselected codon such that it can accept a peptide chain by the action ofthe ribosomal peptidyl transferase when the cognate codon is in thereading position of the ribosome. The peptide chain will be bound insuch a way that the peptide is bound stably and cannot be unattached bythe peptidyl transferase. Preferably, the selected codon is recognizedby hydrogen bonding.

One method for creating a SATA was published in 1973 (Fraser and Rich,PNAS 70:2671 (1973), herein incorporated by reference). This methodinvolves the conversion of a tRNA, or tRNA analog, to a3′-amino-3′-deoxy tRNA. This is accomplished by adding a3′-amino-3′-deoxy adenosine to the end of a native tRNA with tRNAnucleotidyl transferase after removing the native adenosine from it withsnake venom phosphodiesterase. This modified tRNA is then charged withan amino acid by the respective aminoacyl tRNA synthetase (aaRS). Fraserand Rich used an aaRS in which the tRNA is charged on the 3′, ratherthan the 2′, hydroxyl. The amino acid is bound to the tRNA by a stableamide bond rather than the usual labile high-energy ester bond. Thus,when it accepts a peptide from ribosomal peptidyl transferase it willstably hold the peptide and not be able to donate it to anotheracceptor.

In a preferred method, the SATA will be attached to the translatedmessage by a psoralen cross link between the codon and anticodon.Psoralen cross links are preferentially made between sequences thatcontain complementary 5′ pyrimidine-purine 3′ sequences especially UA orTA sequences (Cimino et al., Ann. Rev. Biochem. 54:1151 (1985), hereinincorporated by reference). The codon coding for the SATA, or thelinking codon, can be PYR-PUR-X or X-PYR-PUR, so that several codons maybe used for the linking codon. Conveniently, the stop or nonsense codonshave this configuration. Using a codon that codes for an amino acid mayrequire minor adjustments to the genetic code, which could complicatesome applications. Therefore, in a preferred embodiment, a stop codon isused as the linking codon and the SATA functions as a nonsensesuppressor in that it recognizes the linking codon. One skilled in theart, however, will appreciate that, with appropriate adjustments to thesystem, any codon can be used.

Fraser and Rich did their work in E. coli, but the most effective invitro translation systems are in eukaryotes. The use of prokaryoticsuppressors in eukaryotic translation systems appears to be feasible(Geller and Rich Nature 283:41 (1980); Edwards et al PNAS 88:1153(1991); Hou and Schimmel Biochem 28:6800 (1989), all herein incorporatedby reference). They are primarily limited by the resident aaRS's. Thislimitation is overcome by various embodiments of the present inventionbecause the tRNA or analog can be charged in the prokaryotic system andthen purified according to established methods (Lucas-Lenard and Haenni,PNAS 63:93 (1969), herein incorporated by reference).

In several embodiments of the current invention, acceptor stemmodifications suitable for use in the tRNAs and analogs can be producedby various methods known in the art. Such methods are found in, forexample, Sprinzl and Cramer, Prog. Nuc. Acid Res. 22:1 (1979), hereinincorporated by reference. In an alternative embodiment,“transcriptional tRNA”, i.e. the sequence of the tRNA as it would betranscribed rather than after the post-transcriptional processing, leadsto the atypical and modified bases that are common in tRNAs. Thesetranscriptional tRNAs are capable of functioning as tRNAs (Dabrowski etal., EMBO J. 14: 4872, 1995; and Harrington et al., Biochem. 32: 7617,1993, both herein incorporated by reference). Transcriptional tRNA canbe produced by transcription or can be made by connecting commercial RNAsequences (such as those available from Dharmacon Research Inc.,Boulder, Colo.) together, piece-wise as in FIG. 3, or in somecombination of established methods. For instance, the 5′ phosphate and3′ puromycin are commercially available attached tooligoribonucleotides. These pieces can be connected together using T4DNA ligase, as is well-known in the art (Moore and Sharp, Science 256:992, 1992, herein incorporated by reference). Alternatively, in apreferred embodiment, T4 RNA ligase is used (Romaniuk and Uhlenbeck,Methods in Enzymology 100:52 (1983), herein incorporated by reference).

In several embodiments of the present invention, psoralen ismonoadducted to the SATA by construction of a tRNA from pieces includinga psoralen linked oligonucleotide (FIG. 3) or by monoadduction to anative or modified tRNA or analog (FIG. 4).

In several embodiments, translation will stop when the nascent proteinis attached to the SATA by the peptidyl transferase. When a large numberof ribosomes are in this position the SATA and the mRNA will beconnected with UV light. In a preferred method this will be accomplishedby having a psoralen crosslink formed. Psoralens have a furan side and apyrone side, and they readily intercalate between complementary basepairs in double stranded DNA, RNA, and DNA-RNA hybrids (Cimino et al.,Ann. Rev. Biochem. 54:1151 (1985), herein incorporated by reference).Upon irradiation with UV, preferably in the range of 320 nm to 400 nm,cross linking will take place and leave the staggered pyrimidinescovalently bound. By either forming crosslinks and photo reversing themor by using selected wavelengths, it is possible to form monoadducts,described more fully below. These will be either pyrone sided or furansided monoadducts. Upon further irradiation, the furan sided monoadductscan be covalently crosslinked to complementary base pairs. The pyronesided monoadducts cannot be further crosslinked. The formation of thefuran sided psoralen monoadduct (MAf) is also done according toestablished methods. In a preferred method, the psoralen is attached tothe anticodon of the SATA. However, psoralen can also be attached at theend of the reading frame of the message, as depicted in FIG. 4.

Methods for large scale production of purified MAf on oligonucleotidesare described in the literature (e.g., Speilmann et al., PNAS 89:4514,1992, herein incorporated by reference), as are methods that requireless resources, but have some non-cross-linkable pyrone sided psoralenmonoadduct contamination (e.g., U.S. Pat. No. 4,599,303; Gamper et al.,J. Mol. Biol. 197: 349 (1987); Gamper et al., Photochem. Photobiol.40:29 (1984), both herein incorporated by reference). In severalembodiments of the current invention, psoralen labeling is accomplishedby using either method. In a preferred embodiment, furan sidedmonoadducts will be created using visible light, preferably in the rangeof approximately 400 nm-420 nm, according to the methods described inU.S. Pat. No. 5,462,733 and Gasparro et al., Photochem. Photobiol.57:1007 (1993), both herein incorporated by reference. In one aspect ofthis invention, a SATA with a furan sided monoadduct or monoadductedoligonucleotides for placement on the 3′ end of mRNAs, along with anonadducted SATA are provided as the basis of a kit.

Use of the SATA and the monoadduct in several embodiments of the currentinvention is particularly advantageous for in vitro translation systems.However, one skilled in the art will appreciate that in situ systems canalso be used. Various embodiments of the current invention will beapplicable to any in vitro translation system, including, but notlimited to, rabbit reticulocyte lysate (RLL), wheat germ, E. coli, yeastlysate systems, etc. Many embodiments of the current invention are alsowell-suited for use in hybrid systems where components of differentsystems are combined.

tRNAs aminoacylated on a 3′ amide bond are reported not to combine withthe elongation factor EF-TU which assists in binding to the A site(Sprinzl and Cramer, Prog. Nuc. Acid Res. 22:1 (1979), hereinincorporated by reference). Such modified tRNAs do, however, bind to theA site. This binding of 3′ modified tRNAs can be increased by changingthe Mg⁺⁺ concentration (Chinali et al., Biochem. 13:3001 (1974), hereinincorporated by reference). The appropriate concentrations of and/ormolar ratios of SATA and Mg⁺⁺ can be determined empirically. If theconcentration or A site avidity of SATA is too high, the SATA couldcompete with native tRNAs for non-cognate codons i.e., could functionmuch like puromycin and stall translation. If the concentration or Asite avidity of SATA is too low, the SATA might not effectively competewith the release factors, i.e., it would not act as an effectivenonsense suppressor tRNA. The balance between these can be determinedempirically.

It is also believed that the elongation factor aids in proofreading thecodon-anticodon recognition. The error rate in the absence of elongationfactor and the associated GTP hydrolysis is estimated to be 1 in 100 forcodons one nucleotide away (Voet and Voet, Biochemistry 2^(nd) ed. pp.1000-1002 (1995), John Wiley and Sons, herein incorporated byreference). In a preferred embodiment, UAA is used as the linking codon.For UAA as the linking codon, there are 7 non stop codons which differby one amino acid. This is 7/61 or about 11.5% of the non stop codons.One can estimate the probability of miscoding a given codon as(0.01)(0.115)=1.15×10⁻³ miscodes per codon. Thus, one would expect amiscode about every 870 codons, a frequency which will not substantiallyimpair performance of various methods of the current invention. In analternative embodiment, UAG is used as the linking codon.

In several embodiments, appropriate concentrations of SATA and Mg⁺⁺ areused in the in vitro translation system, e.g. RRL, in the presence ofthe mRNA molecules in the pool, causing translation to cease when theribosome reaches the codon which permits the SATA to accept the peptidechain (the linking codon described above). Within a short time, most ofthe linking codons will be occupied by SATAs within ribosomes. In apreferred embodiment, the system then will be irradiated with UV light,preferably at approximately 320 nm to 400 nm. Nucleic acids aretypically transparent to, i.e. do not absorb, this wavelength range.Upon irradiation, the psoralen monoadduct will convert to a crosslinkconnecting the anticodon and the codon by a stable covalent bond.

In a preferred embodiment, the target mRNA is pre-selected. In anotherembodiment, the target mRNA is artificially produced. In an alternativeembodiment, the target consists of messages native to the system underinvestigation, which may be unknown and/or unidentified. The ability touse unknown and/or unidentified mRNAs is a particular advantage ofseveral embodiments of the current invention.

In several embodiments, once all the nascent proteins are connected totheir cognate mRNAs, the ribosomes are released or denatured.Preferably, this is accomplished by the depletion of Mg⁺⁺ throughdialysis, simple dilution, or chelation. One skilled in the art willunderstand that other methods, including, but not limited to,denaturation by changing the ionic strength, the pH, or the solventsystem can also be used.

In several embodiments of the invention, the selection of cognate pairswill be based upon affinity binding of proteins according to any of avariety of established methods, including, but not limited to, affinitycolumns, immunoprecipitation, and many high throughput screeningprocedures. A variety of ligands may also be used, including, but notlimited to, proteins, nucleic acids, chemical compounds, polymers andmetals. In addition, cell membranes or receptors, or even entire cellsmay be used to bind the cognate pairs. The selection can be positive ornegative. That is, the selected cognate pairs can be those that do bindwell to a ligand or those that do not. For instance, for a protein toaccelerate a thermodynamically favorable reaction, i.e., act as anenzyme for that reaction, it should bind both the substrate and atransition state analog. However, the transition state analog should bebound much more tightly than the substrate. This is described by theequation

$\frac{k_{enzyme}}{k_{\varphi\;{enzyme}}} = \frac{K_{trans}}{K_{subst}}$where the ratio of the rate of the reaction with the enzyme, k_(enzyme),to the rate without, k_(Ψenzyme), is equal to the ratio of the bindingof the transition state to the enzyme K_(trans) over the binding of thesubstrate to the enzyme K_(subst) (Voet and Voet, Biochemistry 2^(nd)ed. p.380, (1995), John Wiley and Sons, herein incorporated byreference).

In a preferred embodiment, proteins which compete poorly for binding tothe substrate but compete well for binding to the transition stateanalog are selected. Operationally, this may be accomplished by takingthe proteins that are easily eluted from a matrix with substrate orsubstrate analog bound to it and are the most difficult to remove frommatrix with transition state analog bound to it. By sequentiallyrepeating this selection and reproducing the proteins throughreplication and translation of the nucleic acid of the cognate pairs, animproved enzyme should evolve. Affinity to one entity and lack ofaffinity to another in the same selection process is used in severalembodiments of the current invention. Selection can also be done by RNAin many embodiments.

Once the selection has identified a population of cognate pairs it maybe convenient to detach the mRNA strand from the SATA to reproduce it.This is not always necessary but can be accomplished by irradiating thepairs with WV, preferably at approximately 313 nm or just below. Thishas been identified as a wave length that will photoreverse the psoralencrosslink to MAf and damage the nucleic acid minimally. The ratio ofphotoreversal to nucleic acid damage is estimated to be 1 photoreversalfor damage to 1 in 600 bases (Cimino et al., Biochem 25:3013 (1986),herein incorporated by reference).

One skilled in the art will appreciate that the mRNAs can be reproducedin many ways, including, but not limited to, by RNA-dependent RNApolymerases or by reverse transcription and PCR. This can take placeusing mRNAs separated from the cognate pairs, e.g., using poly T or polyU to hybridize to the poly A tails of, for instance, native unknownmessages or by leaving the cognate pairs intact and usingoligonucleotide primers that hybridize partially into the reading framefor known messages. Alternatively, commercial kits for rapidamplification of cDNA ends may be used. When this is used to evolveproteins and not just to select them, it would be preferable to sampleat least one amino acid substitution at each position in the protein.

The Replication Threshold

A nominal minimum number of replications for efficient evolution may beestimated using the following formulae. If there is a sequence which isn sequences in length, with a selective improvement r mutations awaywith a mutation rate of p, the probability of generating the selectiveimprovement on replication may be determined as follows:

For r=1, probability of a mutation at the right point, p, times theprobability that it mutated to the right one of the three nucleotidesthat are different from the starting point, ⅓, times the probabilitythat the other n−1 sites remain unmutated, (1−p)(n−r), or

$P_{r} = {\left( \frac{p}{3} \right)^{1}\left( {1 - p} \right)^{({n - 1})}}$where, P=the probability of attaining a given change r mutations away.More generally, for all r values:

$P_{r} = {\left( \frac{p}{3} \right)^{r}\left( {1 - p} \right)^{({n - r})}}$

It is instructive to compare the chances of finding an advantage onemutation away with the chances three mutations away. This is because,given the triplet genetic code, any given codon can only change intonine other codons in one mutation. Indeed, it turns out that no codoncan actually change into nine other amino acid codes in one mutation.The maximum number of amino acids that can be accessed in one mutationis seven amino acids and there are only eight codons of the sixty-fourthat can do this. Most codons have five or six out of nineteen otheramino acids within one mutation. To reach all nineteen amino acids thatare different from the starting one requires, in general, threemutations. These three mutations cannot be sequential since the twointervening ones will not, in general, be selectively advantageous.Therefore we need to use steps that are, at least, three mutations insize (r=3) to use all 20 amino acids.

For a mutation rate of 0.0067, which is that reported for “error-pronePCR”, using a message of 300 nucleotides, which gives a short protein of100 amino acids:P ₃=1.51×10⁻⁹Therefore, one would expect to need a threshold of:

$\frac{1}{1.51 \times 10^{- 9}} = {6.64 \times 10^{8}}$replications at that mutation rate to reasonably expect to reach thenext amino acid that is advantageous. This is not the replication to usesince the binomial expansion shows that over ⅓ of trials (actually about1/e) would not contain the given sequence with selective advantage.

A poisson approximation for large n and small p for a given μ can becalculated so that we can compute the general term when n is, say, ofthe order 10⁹ and p is of the order 10⁻⁹. The general term of theapproximation is:

$\frac{\mu^{\; r}}{{r!}{\mathbb{e}}^{\;\mu}}$

An amplification factor of greater than approximately 6/P ensures thatevolution will progress with the use of all amino acids. This is usefulwhen the production of novel proteins precludes the use of “shuffling”of preexisting proteins.

Limits on Purification

Given a reversible binding where B and C compete for A:

$\begin{matrix}{\left. {AB}\leftrightarrow{A + {B\mspace{59mu} A\; C}}\leftrightarrow{A + C} \right.{k_{B} = {{\frac{\lbrack A\rbrack\lbrack B\rbrack}{\lbrack{AB}\rbrack}\mspace{59mu} k_{C}} = {{\frac{\lbrack A\rbrack\lbrack C\rbrack}{\left\lbrack {A\; C} \right\rbrack}\mspace{56mu}\lbrack B\rbrack} = {k_{B}\frac{\lbrack{AB}\rbrack}{\lbrack A\rbrack}}}}}} & (1) \\{\lbrack C\rbrack = {k_{C}\frac{\left\lbrack {A\; C} \right\rbrack}{\lbrack A\rbrack}}} & (2)\end{matrix}$The total concentrations can be expressed:[B] _(T) =[B]+[AB]  (3)[C] _(T) =[C]+[AC]  (4)Dividing (3) by (4)

$\frac{\lbrack B\rbrack_{T} = {\lbrack B\rbrack + \lbrack{AB}\rbrack}}{\lbrack C\rbrack_{T} = {\lbrack C\rbrack + \lbrack{AC}\rbrack}}$And substituting (1) and (2) for [B] and [C]:

$\frac{\lbrack B\rbrack_{T} = {{k_{B}\left\lbrack \frac{AB}{A} \right\rbrack} + \lbrack{AB}\rbrack}}{\lbrack C\rbrack_{T} = {{k_{C}\left\lbrack \frac{AC}{A} \right\rbrack} + \lbrack{AC}\rbrack}}$Rearranging:

$\frac{\lbrack B\rbrack_{T}}{\lbrack C\rbrack_{T}} = \frac{\lbrack{AB}\rbrack\left( \frac{k_{B} + \lbrack A\rbrack}{\lbrack A\rbrack} \right)}{\lbrack{AC}\rbrack\left( \frac{k_{C} + \lbrack A\rbrack}{\lbrack A\rbrack} \right)}$Canceling the [A]'s in the numerator and denominator:

$\frac{\lbrack B\rbrack_{T}}{\lbrack C\rbrack_{T}} = \frac{\lbrack{AB}\rbrack\left( {k_{B} + \lbrack A\rbrack} \right)}{\lbrack{AC}\rbrack\left( {k_{c} + \lbrack A\rbrack} \right)}$Finally rearranging:

$\frac{\lbrack{AB}\rbrack}{\lbrack{AC}\rbrack} = \frac{\lbrack B\rbrack_{T}\left( {k_{C} + \lbrack A\rbrack} \right)}{\lbrack C\rbrack_{T}\left( {k_{B} + \lbrack A\rbrack} \right)}$$\frac{\left( {k_{C} + \lbrack A\rbrack} \right)}{\left( {k_{B} + \lbrack A\rbrack} \right)}$(Enrichment Factor)

The above factor is termed the “Enrichment Factor”. The ratio of thetotal components is multiplied by this factor to calculate the ratio ofthe bound components, or the enrichment of B over C. The maximumenrichment factor is k_(c)/k_(B), when the [A] is significantly smallerthan k_(c) or k_(B).

The enrichment is limited by the ratio of binding constants. To enrich ascarce protein that is bound 100 times as strongly as its competitors,the ratio of that protein to its competitors is increased by 1 millionwith 3 enrichments. To enrich a protein that only binds twice asstrongly than its competitors, 10 enrichment cycles would gain only anenrichment of ˜1000.

The following Example illustrate various embodiments of the presentinvention and are not intended in any way to limit the invention.

EXAMPLE 1 Production of the Sata

One skilled in the art will understand that the SATA can be produced ina number of different ways. For example, in a preferred embodiment,three fragments (FIG. 1) are purchased from a commercial source (i.e.Dharmacon Research Inc., Boulder, Colo.). Modified bases and a fragment3 with a pre-attached puromycin on its 3′ end and a PO₄ on its 3′ endare included, all of which are available commercially. Three fragmentsare used to facilitate manipulation of the fragment 2 in forming themonoadduct.

Yeast tRNAAla or yeast tRNAPhe is used; however, sequences can be chosenwidely from known tRNA's. Preferably, sequences with only a limitednumber of U's in the portion that corresponds to the fragment 2 areused. Using a sequence with only a few U's is not necessary becausepsoralen preferentially binds 5′UA3′ sequences (Thompson J. F, et alBiochemistry 21:1363, herein incorporated by reference). However, therewould be less doubly adducted product to purify out if such a sequencewas used.

The fragment 2 is used in a helical conformation to induce the psoralento intercalate. Accordingly, a complementary strand is required. RNA orDNA is used, and a sequence, such as poly C to one or both ends, isadded to facilitate separation and removal after monoadduct formation isaccomplished.

The fragment 2 and the cRNA are combined in buffered 50 mM NaClsolution. The Tm is measured by hyperchromicity changes. The twomolecules are re-annealed and incubated for 1 hour with the selectedpsoralen at a temperature ˜10° C. less than the Tm. The psoralen isselected based upon the sequence used. For instance, a relativelyinsoluble psoralen, such as 8 MOP, has a higher sequence stringency butmay need to be replenished. A more soluble psoralen, such as AMT, hasless stringency but will fill most sites. Preferably, HMT is used. If afragment 2 is chosen that contains more non-target U's, a greaterstringency is desired. Decreasing the temperature or increasing ionicstrength by adding Mg⁺⁺ is also used to increase the stringency.

Following incubation, psoralen is irradiated at a wavelength greaterthan approximately 400 nm. The irradiation depends on the wavelengthchosen and the psoralen used. For instance, approximately 419 nm 20-150J/cm2 is preferably used for HMT. This process will result in an almostentirely furan sided monoadduct.

Purification of Monoadduct

The monoadduct is then purified by HPLC as described in Sastry et al, J.Photochem. Photobiol. B Biol. 14:65-79, herein incorporated byreference. The fact that fragment 2 is separate from fragment 3facilitates the purification step because, generally, purification ofmonoadducts≧25 mer is difficult (Spielmann et al. PNAS 89: 4514-4518,herein incorporated by reference).

Ligation of Fragment 2 and 3

The fragment 2 is ligated to the fragment 3 using T4 RNA ligase. Thepuromycin on the 3′ end acts as a protecting group this is done as perRomaniuk and Uhlenbeck, Methods in Enzymology 100:52-59 (1983), hereinincorporated by reference. Joining of fragment 2+3 to the 3′ end offragment 1 is done according to the methods described in Uhlenbeck,Biochemistry 24:2705-2712 (1985), herein incorporated by reference.Fragment 2+3 is 5′ phosphorylated by polynucleotide kinase and the twohalf molecules are annealed.

In an alternative method, significant quantities of furan sidedmonoadducted U will be formed by hybridizing poly UA to itself andirradiating as above. The poly UA will then be enzymatically digested toyield furan sided U which will be protected and incorporated into a tRNAanalog by nucleoside phosphoramidite methods. Other methods of formingthe psoralen monoadducts include the methods described in Gamper et al.,J. Mol. Biol. 197: 349 (1987); Gamper et al., Photochem. Photobiol.40:29, 1984; Sastry et al, J. Photochem. Photobiol. B Biol. 14:65-79;Spielmann et al. PNAS 89:4514-4518, U.S. Pat. No. 4,599,303, all hereinincorporated by reference.

SATAs generated by the methods described above will read UAG (anticodonCUA). Additionally, UAA or UGA will be also be used. In variousembodiments, any message that has the stop codon that is selected as the“linking codon” is used.

EXAMPLE 2 Production of Psoralenated Furan Sided Monoadducts fromCUAGAΨCUGGAGG RNA Fragments

UV Light Exposure Of RNA:DNA Hybrids

Equal volumes of 3 ng/ml RNA:cRNA hybrid segments and of 10 μg/ml HMTboth comprised of 50 mM NaCl are transferred into a new 1.5 ml cappedpolypropylene microcentrifuge tube and incubated at 37° C. for 30minutes in the dark. This was then transferred onto a new clean culturedish. This is positioned in a photochemical reactor (419 nm peakSouthern New England Ultaviolet Co.) at a distance of about 12.5 cm sothat irradiance is ˜6.5 mW/cm2 and irradiated for 60-120 minutes.

Removal of Low Molecular Weight Protoproducts

100 μl of chloroform-isoamyl alcohol (24:1) is pipetted and mixed byvortex. Mixture is centrifuged for 5 minutes at 15000×g in amicrocentrifuge tube. The chloroform-isoamyl alcohol layer is removedwith a micropipet. The chloroform-isoamyl alcohol extraction is repeatedonce again. Clean RNA is precipitated out of the solution.

Alcohol Precipitation

Two volumes (˜1000 μl) ice cold absolute ethanol is added to themixture. The tube is centrifuged for 15 minutes at 15,000×g in amicrocentrifuge. The supernatant is decanted and discarded and theprecipitated RNA is redissolved in 100 μl DEPC treated water thenre-exposed to the RNA+8-MOP.

Isolation of the Psoralentated RNA Fragments Using HPLC

All components, glassware and reagents are prepared so that they areRNAase free. The HPLC is set up with a Dionex DNA PA-100 package column.The psoralenated RNA:DNA hybrid is warmed to 4° C. The psoralenated RNAis applied to HPLC followed by olignucleotide analysis, as described inthe following section entitled “Olignucleotide Analysis by HPLC.” Thecollected fractions will represent:

a) 5′CUAGAΨCUGGAGG3′, (SEQ ID NO:1) where Ψ is pseudouridine b) Furansided (SEQ ID NO:2) 5′CUPsoralenAGAΨCUGGAGG3′ monoadducts c)5′XXXXXCCUCCAGAUCUAGXXXXX3′ (SEQ ID NO:3) d) 5′XXXXXCCUCCAGAUCUP (SEQ IDNO:4) soralenAGXXXXX3′

The fractions are stored at 4° C. in new, RNAase free snappedmicrocentrifuge tubes and stored at −20° C. if more than four weeks ofstorage is required.

Identification of the RNA Fragments Represented by Each Peak FractionCollected by HPLC Using Polyacrylamide Gel Electrophoresis (PAGE)

The electrophoresis unit is set up in a 4° C. refrigerator. A gel isselected with a 2 mm spacer. Each 5 μl of HPLC fraction is diluted to 10μl with Loading Buffer. 10 μl of each diluted fraction is loaded intoappropriately labeled sample wells. The tracking dye is loaded in aseparate lane and electrophoresis is run as described in the followingsection entitled “Polyacrylamide Gel Electrophoresis (PAGE) ofPsoralenated RNA Fragments.” After the electrophoresis run is complete,the electrophoresis is stopped when the tracking dye has reached theedge of the gel. The apparatus is disassembled. The gel-glass panel unitis placed on the UV light box. UV lights are turned on. The RNA bandsare identified. The bands will appear as denser shadows under UVlighting conditions.

Extraction of the RNA From the Gel

Each band is excised with a new sterile and RNAase free scalpel bladeand transferred into a new 1.5 ml snap capped microcentrifuge tube. Eachgel is crushed against the walls of the microcentrifuge tubes with theside of the scalpel blade. A new blade is used for each sample. 1.0 mlof 0.3M sodium acetate is added to each tube and eluted for at least 24hours at 4° C. The eluate is transferred to a new 0.5 ml snap cappedpolypropylene microcentrifuge tube with a micropipet. A new RNAase freepipette tip is used for each tube and the RNA with ethanol isprecipitated out.

Ethanol Precipitation

Two volumes of ice cold ethanol is added to each eluate then centrifugedat 15,000×g for 15 minutes in a microcentrifuge. The supernatants aredischarged and the precipitated RNA is re-dissolved in 100 μl of DEPCtreated DI water. The RNA is stored in the microcentrifuge tubes at 4°C. until needed. The tubes are stored at −20° C. if storage is for morethan two weeks. The following is the assumed order of rate of migrationfor each fragment in order from fastest to slowest:

a) 5′CUAGAΨCUGGAGG3′ b) Furan sided 5′CUPsoralenAGAΨCUGGAGG3′monoadducts. c) 5′XXXXXCCUCCAGAUCUAGXXXXX3′ d)5′XXXXXCCUCCAGAUCUPsoralenAGXXXXX3′

The tubes are labeled containing the remainder of each fraction with thepresumed chemical sequence and stored at −20° C.

Ethanol Precipitation of RNA

RNA oligonucleotide fragments are precipitated, and all glassware iscleaned to remove any traces of RNase as described in the followingsection entitled “Inactivation of RNases on Equipment, Supplies, and inSolutions.” All solutions are stored in RNAase free glassware andintroduction of nucleases is prevented. Absolute ethanol is stored at 0°C. until used. Micropipetors are used to add two volumes of ice coldethanol to nucleic acids that are to be precipitated in microcentrifugetubes. Capped microcentrifuge tubes are placed into the microfuge andspun at 15,000×g for 15 minutes. The supernatant is discarded andprecipitated RNA is re-dissolved in DEPC treated DI-water. RNA is storedat 4° C. in microcentrifuge tubes until ready to use.

Ligation of RNA Fragments 2 and 3

All glassware is cleaned to remove any traces of RNase as described inthe following section entitled “Inactivation of RNases on Equipment,Supplies, and in Solutions.” The following is added to a new 1.5 mlpolypropylene snap capped microcentrifuge tube using a 100-1000 μl pipetand a new sterile pipet tip is used for each solution:

Fragment 2 (3.0 nM) 125.0 μl Fragment 3 (3.0 nM) 125.0 μl Reactionbuffer 250.0 μl RNA T4 ligase (9-12 U/ml) 42 μl Reaction Buffer RNasefree DI-water 90.00 ml Tris-HCl (50 mM) 0.79 g MgCl2 (10 mM) 0.20 g DTT(5 mM) 0.078 g ATP (1 mM) 0.55 g pH to 7.8 with HCL RNase free DI-waterQS to 100.00 ml

The mixture is gently mixed and the RNA is melted by incubating themixture at 16° C. for one hour in a temperature controlled refrigeratedchamber. RNA is precipitated out of the solution immediately after theincubation is completed.

Alcohol Precipitation

Two volumes (˜1000 μl) of ice cold absolute ethanol are added to thereaction mixture. The microcentrifuge tube is placed in amicrocentrifuge at 15,000×g for 15 minutes. The supernate is decantedand discarded and the precipitated RNA is re-dissolve in 100 μl DEPCtreated water. The mixture is electrophoresed as described in thefollowing section entitled “Polyacrylamide Gel Electrophoresis (PAGE) ofPsoralenated RNA Fragments.” The following is the assumed order of rateof migration for each fragment in order from fastest to slowest:

a) Frag. 2 5′CUAGAΨCUGGAGG3′-OH Psoralen b) Frag. 35′UCCUGUGTΨCGAUCCACAGAAU (SEQ ID NO:5) UCGCACC-Puromycin c) Frag 2 + 35′CUAGAYCUGGAGGUCCUGUGTΨ (SEQ ID NO:6) CGAUCCACAGAAUUCGCACC PuromycinPsoralen

Each fraction is isolated by UV shadowing, the bands are cut out, theRNAs are eluted from the gels and the RNA elute is precipitated out asdescribed in the following section entitled “Polyacrylamide GelElectrophoresis (PAGE) of Psoralenated RNA Fragments.” The ligationprocedure is repeated with any residual unligated fragment 2 and 3fractions. The ligated fractions 2 and 3 are pooled and stored in asmall volume of RNase free DI-water at 4° C.

Ligation of RNA Fragment 1 with Fragment 2+3

All glassware is cleaned to remove any traces of RNase as described inthe following section entitled “Inactivation of RNases on Equipment,Supplies, and in Solutions.” The following is added to a new 1.5 mlpolypropylene snap capped microcentrifuge tube. A 100-1000 μl pipet andnew tip is used for each solution:

Fragment 2 + 3 (3.0 nM) 125.0 μl Reaction buffer 250.0 μl T4Polynucleotide Kinase(5-10 U/ml) 1.7 μl Reaction Buffer RNase freeDI-water 90.00 ml Tris-HCl (40 mM) 0.63 g MgCl2 (10 mM) 0.20 g DTT (5mM) 0.08 g ATP (1 mM) 0.006 g pH to 7.8 with HCL RNase free DI-water QSto 100.00 ml

The RNA is gently mixed then melted by heating the mixture to 70° C. for5 minutes in a heating block. The mixture is cooled to room temperatureover a two hour period and the RNAs is allowed to anneal in a tRNAconfiguration. The RNA is precipitated out of the solution.

Alcohol Precipitation

Two volumes (˜1000 μl) if ice cold absolute ethanol are added to thereaction mixture. The microcentrifuge tube is placed in amicrocentrifuge at 15,000×g for 15 minutes. The supernate is decantedand discarded and the precipitated RNA is re-dissolved in 100 μl DEPCtreated water. The mixture is electrophoresed as described in thefollowing section entitled “Polyacrylamide Gel Electrophoresis (PAGE) ofPsoralenated RNA Fragments.” The following is the assumed order of rateof migration for each fragment in order from fastest to slowest:

a) Frag. 1 5′GCGGAUUUAGCUCAGDD GGGAGAGCGCCAGACU3′ b) Frag 2 + 35′CUAGAYCUGGAGGUCCU GUGTΨCGAUCCACAGAAUU CGCACCPuromycin Psoralen c)Frag. 1 + 2 + 3 5′GCGGAUUUAGCUCAGDD (SEQ ID NO:7) GGGAGAGCGCCAGACUCUAGAΨCUGGAGGUC . . . CUGUGTΨCGAUCCACAGAA UUCGCACCPuromycin Psoralen

Each fraction is isolated by UV shadowing, the bands are cut out, theRNAs are eluted from the gels and the RNA elute is precipitated out asdescribed in the following section entitled “Polyacrylamide GelElectrophoresis (PAGE) of Psoralenated RNA Fragments.” The ligationprocedure is repeated with the unligated Fragment 1 and the 2+3Fraction. The ligated fractions 2+3 are pooled and stored in a smallvolume of RNase free DI-water at 4° C.

Final RNA Ligation The following is added to a new 1.5ml polypropylenesnap capped microcentrifuge tube. A 100-1000 μl pipet and new tip isused for each solution:

Fragment 1 + 2 + 3 (3.0 nM) 250 μl reaction buffer 250 μl RNA T4 ligase(44 μg/ml) 22 μg

The mixture is incubated at 17° C. in a temperature controlledrefrigerator for 4.7 hours. Immediately after the incubation the tRNA isprecipitated out as described in step 6.2 above and the tRNA is isolatedby electrophoresis as described in the following section entitled“Polyacrylamide Gel Electrophoresis (PAGE) of Psoralenated RNAFragments.” The tRNA is pooled in a small volume of RNase free water andstored at 4° C. for up to two weeks or stored at −20° C. for periodslonger than two weeks.

Polyacrylamide Gel Electrophoresis (PAGE) of Psoralenated RNA Fragments

Acrylamide Gel Preparation

All reagents and glassware must be RNAase free as described in thefollowing section entitled “Inactivation of RNases on Equipment,Supplies, and in Solutions.” The gel apparatus is assembled to produce a4 mm thick by 20 cm×42 cm square gel. 29 parts acrylamide with 1 partammonium crosslinker are mixed at room temperature with the appropriateamount of acrylamide solution in an RNAase free, thick walled Erlenmeyerflask.

Acrylamide Solution urea (7M) 420.42 g TBE (1X) QS to 1 L 5X TBE 0.455 MTris-HCl 53.9 g 10 mM EDTA 20 ml of 0.5 M RNAase free DI water 900 ml pHwith boric acid to pH 9 QS with RNAase free DI water to 1 L

The mixture is degassed with vacuum pressure for one minute. Theappropriate amount of TEMED is added, mixed gently, and then the gelmixture is poured between the glass plates to within 0.5 cm of the top.The comb is immediately inserted between the glass sheets and into thegel mixture. An RNAase free gel comb is used. The comb should producewells for a 5 mm wide dye lane and 135 mm sample lanes. The gel isallowed to polymerize for about 30-40 minutes then the comb is carefullyremoved. The sample wells are rinsed out with a running buffer using amicropipet with a new pipet tip. The wells are then filled with runningbuffer.

Sample Preparation

An aliquot of the sample is suspended in loading buffer in a snap cappedmicrocentrifuge tube and vortex mixed. Indicator dye is not added to thesample.

Loading Buffer Urea (7M) 420.42 g Tris HCl (50 mM) 7.85 g QS with RNAasefree D-H2O to 1 LElectrophoresis Run

The maximum volume of RNA/loading buffer solution is loaded into the 135mm sample wells and the appropriate volume of tracking dye in 5 mmtracking lane. The samples are electrophoresed in a 5° C. refrigerator.The electrophoresis is stopped when the tracking dye has reached theedge of the gel. Disassemble the apparatus. Glass panels are not removedthe from the gel. The gel-glass panel unit is placed on a UV light box.With UV filtering goggles in place, the UV lights are turned on. The RNAbands are identified. They appear as denser shadows under UV lightingconditions. The RNA is extracted from the gel. Each bands is excisedwith a new sterile and RNAase free scalpel blade and each band istransferred into a new 1.5 ml snap capped microcentrifuge tube. Each gelis crushed against the walls of the microcentrifuge tubes with the sideof the scalpel blade. A new blade is used for each sample. 1.0 ml of0.3M sodium acetate is added to each tube and eluted for at least 24hours at 4° C. The eluate is transferred to a new 0.5 ml snap cappedpolypropylene microcentrifuge tubes with a micropipet with a new RNAasefree pipet tip for each tube. Two volumes of ice cold ethanol is addedto each eluate, then centrifuged at 15,000×g for 15 minutes in amicrocentrifuge. The supernatants are discarded and the precipitated RNAis redissolved in 100 μl of DEPC treated DI water. The RNA is stored inthe microcentrifuge tubes at 4° C. until needed.

Olignucleotide Analysis by HPLC

HPLC purification of the RNA oligonucleotides is best effected usinganion exchange chromatography. Either the 2′-protected or 2′-deprotectedforms can be chromatographed. The 2′-protected form offers the advantageof minimizing secondary structure effects and provides resistance tonucleases. If the RNA is fully deprotected, sterile conditions arerequired during purification.

Deprotection of 2′-Orthoester Protected RNA

The tubes are centrifuged at 15,000×g for 30 seconds or until the RNApellet is at the bottom. 400 μl of pH 3.8 deprotection buffer is addedto each tube of RNA.

Deprotection Buffer

Acetic acid (100 mM) is adjusted to pH 3.8 withtetramethylethylenediamine (TEMED). The pellet is completely dissolvedin the buffer by drawing in and out of a pipette. The tubes are vortexedfor 10 seconds and centrifuged at 15,000×g. The tubes are incubated in a60° C. water bath for 30 minutes. The samples are lyophilized beforeuse.

HPLC Column Conditions

A 4×250 mm column (DNAPAC PA, No. 043010) packed with Dionex(800)-DIONEX-0 (346-6390), with a capacity of 40 optical density units(ODU) at 260 nm is installed. The column temperature is set to 54° C.The injection volume is adjusted such that 5 μl produces approximately0.20 ODU.

Elution Buffers

Condition Buffer A Buffer B Sodium perchlorate (5 mM) 2.8 g (300 mM)168.0 g Tris-HCl 2.4 g 2.4 g Acetonitrile (2%) 80.0 ml 80.0 ml DI Water3900 ml 900 ml Adjusted pH 8.0 with HCL 8.0 with HCL q.s. 4000 ml 4000mlHPLC Gradient

A 30% to 60% gradient of Buffer B for oligos 17-32 base pairs long isprovided:

Time Flow (minutes) (ml/min) % A % B Curve 0 1.5 100 0 * 1 1.5 100 0 6 31.5  70* 30* 6 15 1.5  40* 60* 6 15.5 2.5  0 100  6 17 2.5  0 100  617.25 2.5 100 0 6 23 2.5 100 0 6 23.1 1.5 100 0 6 24 1.5 100 0 6 25 0.1100 0 6 *% values that can be changed to modify the gradient. Typicalgradients are 0-30%, 20-50%, 30-60%, and 40-70% of Buffer B.Gradient Selection

The gradient is selected based upon the number of bases, as follows:

Number of bases Gradient 0-5  0-30  6-10 10-40 11-16 20-50 17-32 30-6033-50 40-70 >50 50-80

After HPLC, the target samples are collected and the RNA concentrationis determined with a spectrophotometer at 260 nm. The samples are storedat −70° C.

Inactivation of RNases on Equipment, Supplies, and in Solutions

Glassware is treated by baking at 180° C. for at least 8 hours.Plasticware is treated by rinsing with chloroform. Alternatively, allitems are soaked in 0.1% DEPC.

Treatment with 0.1% DEPC

0.1% DEPC is prepared. DI water is filtered through a 0.2 μM membranefilter. The water is autoclaved at 15 psi for 15 minutes on a liquidcycle. 1.0 g (wt/v) DEPC/liter of sterile filtered water is added.

Glass and Plasticware

All glass and plasticware is submerged in 0.1% DEPC for two hours at 37°C. The glassware is rinsed at least 5× with sterile DI water. Theglassware is heated to 100° C. for 15 minutes or autoclaved for 15minutes at 15 psi on a liquid cycle.

Electrophoresis Tanks Used for Electrophoresis of RNA

Tanks are washed with detergent, rinsed with water then ethanol and airdried. The tank is filled with 3% (v/v) hydrogen peroxide (30ml/L) andleft standing for 10 minutes at room temperature. The tank is rinsed atleast 5 times with DEPC treated water.

Solutions

All solutions are made using Rnase free glassware, plastic ware,autoclaved water, chemicals reserved for work with RNA and RNase freespatulas. Disposable gloves are used. When possible, the solutions aretreated with 0.1% DEPC for at least 12 hours at 37° C. and then heatedto 100° C. for 15 minutes or autoclaved for 15 minutes at 15 psi on aliquid cycle.

RNA Translation

2 μl of gastroinhibitory peptide (GIP) mRNA at a concentration of 20μl/ml is placed in a 250 μl snapcap polypropylene microcentrifuge tube.35 μl of rabbit reticulocyte lysate (available commercially fromPromega) is added. 1 μl of amino acid mixture which does not containmethionine (available commercially from Promega) is added. 1 μl of 35Smethione or unlabeled methionine is added. Optionally, 2 ml ofluciferase may be added to some tubes to serve as a control.

SATA is added to the experimental tubes. Control tubes which do notcontain SATA are also prepared. The quantity of SATA used isapproximately between 0.1 μg to 500 μg, preferably between 0.5 μg to 50μg. 1 μl of Rnasin at 40 units/ml is added. Nuclease free water is addedto make a total volume of 50 μl.

For proteins greater than approximately 150 amino acids, the amount oftRNA may need to be supplemented. For example, approximately 10-200 μgof tRNA may be added. In general, the quantity of the SATA should behigh enough to effectively suppress stop or pseudo stop codons. Thequantity of the native tRNA must be high enough to out compete the SATAwhich does not undergo dynamic proofreading under the action ofelongation factors.

Each tube is immediately capped, parafilmed and incubated for thetranslation reactions at 30° C. for 90 minutes. The contents of eachreaction tube is transferred into a 50 μl quartz capillary tube bycapillary action. The SATA is crosslinked with mRNA by illuminating thecontents of each tube with 2-10 J/cm2˜350 nm wavelength light, as perGasparro et al. (Photochem. Photobiol. 57:1007 (1993), hereinincorporated by reference). Following photocrosslinking, the contents ofeach tube is transferred into a new snapcap microfuge tube. Theribosomes are dissociated by chelating the calcium cations by adding 2μl of 10 mM EDTA to each tube. Between each step. each tube is gentlymixed by stirring each component with a pipette tip upon addition.

The optimal RNA for a translation is determined prior to performingdefinitive experiments. Serial dilutions may be required to find theoptimal concentration of mRNA between 5-20 μg/ml.

Reagent 1 2 3 4 Rabbit reticulocyte lysate (35 μl) + + + + Amino acidmixture minus + + + + methionine (1 μl of 1 mM) ³⁵S Methionine (1 μlof + − − + 1,200 Ci/mmol) Methionine (unlabeled) − + + − GIP mRNA (2 μlof 20 μg/ml) + − − − ³²P GIP mRNA (2 μl of 20 μg/ml) − + + − Rnasin (1μl of 40 U/μl) + + + + SATA − − − Water, nuclease free (q.s. to 50μl) + + + +

SDS-Page electrophoresis is performed on each sample, as describedabove. Autoradiography on the gel is performed, as described by Sambrooket. al., Molecular Cloning, A Laboratory Manual, 2^(nd) ed., ColdspringHarbor Press (1989), herein incorporated by reference.

While a number of preferred embodiments of the current invention andvariations thereof have been described in detail, other modificationsand methods of use will be readily apparent to those of skill in theart. Accordingly, it should be understood that various applications,modifications and substitutions may be made without departing from thespirit of the invention or the scope of the claims.

1. A tRNA analogue, comprising: (a) a tRNA; (b) an amino acid moietywhich acts as an acceptor substrate, but not as a donor substrate,during translation; and (c) a psoralen near or within the anticodonstem-loop that can mediate the stable coupling of the tRNA analogue tomRNA.
 2. A tRNA analogue which is a tRNA in which the 3′ terminalnucleotide is replaced by puromycin and in which the anticodon loopcomprises a psoralen that can mediate coupling of the tRNA analogue tomRNA.